A validated method for analyzing polyunsaturated free fatty acids from dried blood spots using LC–MS/MS

• Published in: Prostaglandins, leukotrienes and essential fatty acids Vol. 125; pp. 1 - 7
• Main Authors: Hewawasam, Erandi, Liu, Ge, Jeffery, David W., Muhlhausler, Beverly S., Gibson, Robert A.
• Format: Journal Article
• Language: English
• Published: Scotland Elsevier Ltd 01.10.2017
• Subjects: Analytical method validation; Chromatography, Liquid - methods; Dried blood spot; Dried Blood Spot Testing - methods; Fatty Acids, Nonesterified - analysis; Fatty Acids, Omega-3 - analysis; Fatty Acids, Omega-6 - analysis; Fatty Acids, Unsaturated - analysis; Free fatty acids; Humans; Polyunsaturated fatty acids; Reproducibility of Results; Tandem Mass Spectrometry - methods; AA; LOD; PUFA–FFA; CE; Polyunsaturated fatty acids; Dried blood spot; MRM; IS; DP; BHT; LOQ; Free fatty acids; Analytical method validation; and CXP; AR; FAME; DPA; LA; ALA; EPA; TLC–GC; SEM; DHA
• ISSN: 0952-3278; 1532-2823; 1532-2823
• DOI: 10.1016/j.plefa.2017.08.010

Omega–3 and omega–6 polyunsaturated free fatty acids (PUFA–FFA) are precursors to potent downstream lipid mediators that are regulators of inflammation. We describe the development and validation of a novel and sensitive method for quantification of individual PUFA–FFA in a dried blood spot using liquid chromatography tandem mass spectrometry (LC–MS/MS). Lipids were extracted from dried blood spot and six individual PUFA–FFA were quantified by LC–MS/MS using stable isotope dilution analysis with deuterated internal standards. PUFA–FFA concentrations in blood samples from 30 subjects were measured using the new method and compared to the traditional approach of thin layer chromatography followed by gas chromatography with flame ionization detection (TLC–GC). Responses for each PUFA–FFA were linear throughout a range of concentrations expected in clinical samples. Intra–day and inter–day variations for all PUFA–FFA were ≤ 16%. The concentrations of all PUFA–FFA measured by LC–MS/MS were positively correlated with measures of the same PUFA–FFA obtained by a traditional TLC–GC method. This novel method for the quantification of PUFA–FFA extracted from dried blood is sensitive and precise, and accurately measures levels of biologically important PUFA–FFA in blood. •We describe a new method to measure polyunsaturates in the free fatty acid pool from dried blood spots using LC-MS/MS.•Method is precise, reproducible, accurate and correlates well with traditional approaches.•Our new method has potential to be applied to large clinical trials or population studies.